X radiation up-regulates the occurrence and the multiplicity of invasive carcinomas in the intestinal tract of Apc(min/+) mice

X rays are well known to cause genetic damage and to induce many types of carcinomas in humans. The Apc(min/+) mouse, an animal model for human familial adenomatous polyposis (FAP), contains a truncating mutation in the APC gene and spontaneously develops intestinal adenomas. To elucidate the role of X rays in the development of intestinal tumors, we examined the promotion of carcinogenesis in X-irradiated Apc(min/+) mice. Forty out of 77 (52%) X-irradiated Apc(min/+) mice developed adenocarcinomas that invaded the proprial muscle layer of the small intestine; 24 of 44 (55%) were in males, and 16 of 33 (49%) were in females. In contrast, invasive carcinomas were detected in the small intestines of only 13 of 64 (20%) nonirradiated Apc(min/+) mice; nine of 32 (28%) were in males and four of 32 (13%) were in females. These differences between X-irradiated and nonirradiated Apc(min/+) mice in the occurrence of invasive intestinal carcinomas were statistically significant (P < 0.05 for males, P < 0.005 for females). In wild-type mice, invasive carcinomas were not detected in either X-irradiated or nonirradiated mice. Apc(min/+) mice had many polyps in the large intestine with or without X irradiation; there was no difference in the number of polyps between the two groups. Also, invasive carcinomas were not detected in the large intestine with or without irradiation. The occurrence of mammary tumors, which was observed in Apc(min/+) mice, was found to be increased in irradiated Apc(min/+) mice (P < 0.01). Apc(min/+) mice had many polyps in the small and large intestines with or without X irradiation. X-irradiated Apc(min/+) mice had highly invasive carcinomas in the small intestine with multiplicities associated with invasiveness. Our results suggest that X radiation may promote the invasive activity of intestinal tumors in Apc(min/+) mice.     

Distribution and variable expression of secretory pathway protein reticulocalbin in normal human organs and non-neoplastic pathological conditions.

Reticulocalbin (RCN) is one member of the Ca(2+)-binding proteins in the secretory pathway and is localized in the endoplasmic reticulum. RCN may play a role in the normal behavior and life of cells, although its detailed role remains unknown. Overexpression of RCN may also play a role in tumorigenesis, tumor invasion, and drug resistance. The new antibody for human RCN is used in the distribution of RCN in normal human organs of fetuses and adults with or without inflammation. Immunohistochemical examination demonstrated a broad distribution of RCN in various organs of fetuses and adults, predominantly in the endocrine and exocrine organs. However, RCN expression was heterogeneous in each constituent cell of some organs. Among non-epithelial organs, vascular endothelial cells, testicular germ cells, neurons, and follicular dendritic cells showed strong staining. Plasma cells were the only RCN-positive cells among hematopoietic and lymphoid cells. In inflammatory conditions, RCN expression was enhanced in both epithelial and non-epithelial cells. Heterogeneous expression of RCN indicates that the amount of RCN needed for cell behavior and life may be variable, depending on each cell type and, therefore, RCN may be helpful in establishing the cell origin of neoplasms in some organs. However, further study is needed to establish the significance of RCN in tumorigenesis and in some peculiar features of neoplasms.     

Development of an apparatus for rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in chondroitin sulfate-type proteoglycans

An apparatus, AutoGlycoCutter (AGC), was developed as a tool for rapid release of O-linked-type glycans under alkaline conditions. This system allowed rapid release of oligosaccharides at the glycosaminoglycan-protein linkage region in proteoglycans (PGs). After digestion of PGs with chondroitinase ABC, the oligosaccharides at the linkage region were successfully released from the protein core by AGC within 3 min. The reducing ends of the released oligosaccharides were labeled with 2-aminobenzoic acid and analyzed by a combination of capillary electrophoresis (CE) and matrix-assisted laser desorption time-of-flight mass spectrometry. In addition, the unsaturated disaccharides produced by chondroitinase ABC derived from the outer parts of the glycans were labeled with 2-aminoacridone and analyzed by CE to determine the disaccharide compositions. We evaluated AGC as a method for structural analysis of glycosaminoglycans in some chondroitin-sulfate-type PGs (urinary trypsin inhibitor, bovine nasal cartilage PG, bovine aggrecan, bovine decorin, and bovine biglycan). Recoveries of the released oligosaccharides were 57-73% for all PGs tested in the present study. In particular, we emphasize that the use of AGC achieved ca. 1000-fold rapid release of O-glycans compared with the conventional method.     

Changes in urinary dinor dihydro F(2)-isoprostane metabolite concentrations, a marker of oxidative stress, during and following asthma exacerbations

To investigate changes in oxidant stress during and following acute asthma exacerbations, this study measured 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP (F(2)-IsoP-M), the major urinary metabolite of 15-F(2t)-IsoP, in eight asthmatic adults, during and following an asthma hospitalization. F(2)-IsoP-M concentrations at admission and follow-up were significantly higher than discharge (admission median: 4.12 ng/Cr mg, range 1.89-7.8; follow-up: 2.47 ng/Cr mg (1.56-6.86); discharge: 1.42 ng/Cr mg (0.7-4.44); both p<0.01), but not significantly different between admission and follow-up. F(2)-IsoP-M concentrations at follow-up were higher than a control group with stable asthma (0.68 ng/Cr mg (0.31-1.5), p=0.0008). In conclusion, asthma exacerbations requiring hospitalization are associated with 6-fold higher urinary F(2)-IsoP-M concentrations compared to stable asthmatics. F(2)-IsoP-M concentrations decreased significantly during hospitalization, but significant elevations 3 months following hospitalization suggest ongoing oxidative stress despite clinical improvement. Urinary F(2)-IsoP-M may be a clinically useful, simple non-invasive systemic measure of oxidative stress in asthmatics, providing information not captured by spirometry or symptoms.     

In situ observation of a cell adhesion and metabolism using surface infrared spectroscopy

In this study, we report on an in situ monitoring system of living cultured cells using infrared absorption spectroscopy in the geometry of multiple internal reflections (MIR-IRAS). In order to observe living cultured cells, the temperature in the sample chamber of a FT-IR spectrometer was maintained at 37 °C and a humidified gas mixture containing 5% CO₂ was introduced into the sample chamber. Human breast cell line MCF-7 cultured on Si MIR prisms were placed in the sample chamber and infrared spectra of MCF-7 cells were collected for 5 h. It was found that the adhesion and metabolism of MCF-7 cells could be monitored by the absorption intensity of amide-II protein band (1,545 cm⁻¹) and also by the absorption intensities of CH _x bands (2,700–3,100 cm⁻¹). These results suggest that our system is useful for a nondestructive and non-label monitoring of cell viability. Our method based on infrared absorption spectroscopy has a potential for bioscreening application.     

Exogenous cytosine deaminase gene expression in Bifidobacterium breve I-53-8w for tumor-targeting enzyme/prodrug therapy

Bifidobacteria are nonpathogenic, anaerobic domestic bacteria with health-promoting properties for the host. In our previous study, Bifidobacterium longum (B. longum) were found to be localized selectively and to proliferate within solid tumors after systemic application. Additionally, B. longum transformed by shuttle-plasmid including the cytosine deaminase (CD) gene expressed active CD, converted the prodrug 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). We also demonstrated antitumor efficacy with a transformant of B. longum in rats. In this study, we found that Bifidobacterium breve (B. breve), the smallest species of human-derived bifidobacterium, expressed the exogenous transgene (CD), that CD enzymatic activity in the transformant of B. breve was much higher, and that the segregational stability of the plasmid was greater than that of B. longum. Thus, numerous transformants of B. breve were detected solely in the tumors after systemic administration. We consider the transformant of B. breve to be more beneficial in our enzyme/prodrug therapy.     

Repellent from traditional Chinese medicine, Periploca sepium Bunge

By using a new bioassay-guided method, 2-hydroxy-4-methoxybenzaldehyde isolated from the root bark of Periploca sepium, a traditional Chinese medicine, showed repellent activity against the olive weevil (Dyscerus perforatus) at 62.5, 125, 250 and 500 microg/disc, respectively. In addition, it also exhibited antinematodal activity against Bursaphelenchus xylophilus at a minimum effective dose of 200 microg/ball. The three related compounds obtained were also evaluated for the above-mentioned bioactivities.     

Involvement of vasa homolog in germline recruitment from coelomic stem cells in budding tunicates

We investigated the mechanism by which germline cells are recruited in every asexual reproductive cycle of the budding tunicate Polyandrocarpa misakiensis using a vasa homolog (PmVas) as the germline-specific probe. A presumptive gonad of Polyandrocarpa arose as a loose cell aggregate in the ventral hemocoel of a 1-week-old developing zooid. It developed into a compact clump of cells and then separated into two lobes, each differentiating into the ovary and the testis. The ovarian tube that was formed at the bottom of the ovary embedded the oogonia and juvenile oocytes, forming the germinal epithelium. PmVas was expressed strongly by loose cell aggregates, compact clumps, and peripheral germ cells in the testis and germinal epithelium. No signals were detected in growing buds and less than 1-week-old zooids, indicating that germ cells arise de novo in developing zooids of P. misakiensis. Cells of the loose cell aggregates were 5–6 μm in diameter. They looked like undifferentiated hemoblasts in the hemocoel. To examine the involvement of PmVas in the germline recruitment at postembryonic stages, both growing buds and 1-week-old developing zooids were soaked with double-stranded PmVas RNA. The growing buds developed into fertile zooids expressing PmVas, whereas the 1-week-old zooids developed into sterile zooids that did not express PmVas. In controls (1-week-old zooids) soaked with double-stranded lacZ RNA, the gonad developed normally. These results strongly suggest that in P. misakiensis, PmVas plays a decisive role in switching from coelomic stem cells to germ cells.     

Reexamination of Chlorophyllase Function Implies Its Involvement in Defense against Chewing Herbivores

Chlorophyllase (CLH) is a common plant enzyme that catalyzes the hydrolysis of chlorophyll to form chlorophyllide, a more hydrophilic derivative. For more than a century, the biological role of CLH has been controversial, although this enzyme has been often considered to catalyze chlorophyll catabolism during stress-induced chlorophyll breakdown. In this study, we found that the absence of CLH does not affect chlorophyll breakdown in intact leaf tissue in the absence or the presence of methyl-jasmonate, which is known to enhance stress-induced chlorophyll breakdown. Fractionation of cellular membranes shows that Arabidopsis (Arabidopsis thaliana) CLH is located in the endoplasmic reticulum and the tonoplast of intact plant cells. These results indicate that CLH is not involved in endogenous chlorophyll catabolism. Instead, we found that CLH promotes chlorophyllide formation upon disruption of leaf cells, or when it is artificially mistargeted to the chloroplast. These results indicate that CLH is responsible for chlorophyllide formation after the collapse of cells, which led us to hypothesize that chlorophyllide formation might be a process of defense against chewing herbivores. We found that Arabidopsis leaves with genetically enhanced CLH activity exhibit toxicity when fed to Spodoptera litura larvae, an insect herbivore. In addition, purified chlorophyllide partially suppresses the growth of the larvae. Taken together, these results support the presence of a unique binary defense system against insect herbivores involving chlorophyll and CLH. Potential mechanisms of chlorophyllide action for defense are discussed.Plants have evolved both constitutive and inducible defense mechanisms against herbivores. Constitutive mechanisms include structural defenses (e.g. spines and trichomes) and specific chemical compounds. Constitutive defense mechanisms provide immediate protection against herbivore attacks, although they represent an energy investment by the plant regardless of whether herbivory occurs or not (Mauricio, 1998; Bekaert et al., 2012). By contrast, inducible defense mechanisms do not require an up-front energy cost, although such mechanisms may not be as immediate as constitutive ones when herbivore feeding occurs (Windram et al., 2012). Accordingly, plants exhibit both constitutive and inducible defense mechanisms against herbivory to balance the speed and cost of response. In this regard, it is plausible that the recruitment of abundant primary metabolites for defensive purposes might represent a substantial benefit to plants, providing both a swift and economical defense function.Toxic chemical compounds form an essential part in both constitutive and inducible defense mechanisms. However, these compounds are potentially a double-edged sword for plants, in a sense that they might pose toxic effects for both plants and herbivores. Plants have evolved an intricate binary system that prevents autointoxication by their own chemical compounds. Specifically, a toxic substance is stored in its inactive form and is spatially isolated from specific activating enzymes. These enzymes activate the substance when cells are disrupted by chewing herbivores (Saunders and Conn, 1978; Thayer and Conn, 1981; Morant et al., 2008). One of the most extensively studied binary defense systems is the glucosinolate/myrosinase system, in which the glucosinolate substrate and their hydrolyzing enzyme, a thioglucosidase myrosinase, are compartmentalized. Upon tissue damage, both the substrate and the enzyme come into contact to produce unstable aglycones, and various toxic compounds are then spontaneously produced (Bones and Rossiter, 1996). Another well-known example of the binary system is comprised of cyanogenic glucosides and β-glucosidase (Vetter, 2000; Mithöfer and Boland, 2012). In this system, nontoxic cyanogenic glycoside compounds are stored in the vacuole, whereas, the related glycosidase is localized in the cytoplasm. Upon cell destruction by chewing herbivores, the cyanogenic glycosides are hydrolyzed by glycosidase to yield unstable cyanohydrin that is either spontaneously or enzymatically converted into toxic hydrogen cyanide and a ketone or an aldehyde. Because the binary defense system is efficient and effective, a use of ubiquitous compounds for such systems would provide further benefits for plants.Tetrapyrrole compounds, in particular heme and chlorophyll, are abundant in plant cells. Despite their significant roles in various biological processes including photosynthesis and respiration, many tetrapyrroles are highly toxic to plant and animal cells, if present in excess amounts (Kruse et al., 1995; Meskauskiene et al., 2001). Their photodynamic properties can cause the generation of reactive oxygen species upon illumination, resulting in cell injury or direct cell death. For example, Tapper et al. (1975) showed that a tetrapyrrole compound (pheophorbide a), which is readily converted from dietary chlorophyll through the loss of magnesium and phytol, reduces the growth and survival rates of young albino rats through its photodynamic property. More recently, Jonker et al. (2002) demonstrated that dietary-derived pheophorbide a causes severe damages on the skin of mutant mice that lack a transporter to excrete pheophorbide a from cells. These studies indicate that incorporation of an excessive amount of tetrapyrrole compounds can induce photosensitization in animals. Previous studies also showed that tetrapyrroles have illumination-independent deleterious effects on insects. For example, pheophorbide a affected the assimilation of the plant sterols to synthesize developmental hormones of insects by inhibiting the activity of a key enzyme, cholesterol acyltransferase (Song et al., 2002). Moreover, some tetrapyrroles, including pheophorbide a, have been suggested to induce illumination-independent cell death in plants as well by an unknown mechanism (Hirashima et al., 2009). It is proposed that organisms use the toxicity of tetrapyrroles for their defense systems. The larvae of tortoise beetle (Chelymorpha alternans) even utilize pheophorbide a as a powerful deterrent in the fecal shield to protect themselves from their predators (Vencl et al., 2009). Kariola et al. (2005) suggested that a chlorophyll derivative, chlorophyllide, is involved in the defense against fungi, based on their observations that down-regulation of a chlorophyll-hydrolyzing enzyme, chlorophyllase (CLH), results in increased susceptibility of Arabidopsis (Arabidopsis thaliana) plants to the necrotrophic fungus Alternaria brassicicola.In this study, we examined the possibility that plants use tetrapyrroles for defense against herbivores by analyzing CLH, a well-known hydrolase common in plants. Chlorophyll consists of a tetrapyrrolic macrocycle and a hydrophobic phytol side chain (Fig. 1). Phytol hydrolysis results in the formation of chlorophyllide (Fig. 1), a less hydrophobic chlorophyll derivative, which has photochemical properties similar to chlorophyll. Two different plant enzymes are known to catalyze the cleavage of phytol, pheophytinase (PPH) and CLH. PPH is a chloroplast-located enzyme that specifically catalyzes the removal of phytol from Mg-free chlorophyll catabolites (Schelbert et al., 2009). This enzyme was only recently discovered and has been shown to be responsible for chlorophyll degradation during leaf senescence. By contrast, CLH has a broader substrate specificity and removes the side chain from chlorophyll or other chlorophyll derivatives (McFeeters et al., 1971). CLH activity was first reported in leaf extracts in 1913 (Willstätter and Stoll, 1913), but despite a century of research, in vivo function and intracellular localization of this enzyme remained controversial. Some reports have indicated CLH to localize to chloroplasts (Azoulay Shemer et al., 2008; Azoulay-Shemer et al., 2011), while Schenk et al. (2007), by examining the intracellular localization of transiently expressed CLH-GFP fusions, proposed Arabidopsis CLH to localize outside the chloroplast. Schenk et al. (2007) also reported that the lack of CLH does not affect chlorophyll degradation during leaf senescence. However, it remains possible that CLH is specifically involved in chlorophyll degradation in response to stresses that activate jasmonate signaling, such as wounding or pathogen attack. This hypothesis is based on the observation that the expression of a CLH gene was highest when methyl-jasmonate (MeJA; a derivative of jasmonic acid) was applied to Arabidopsis plants (Tsuchiya et al., 1999).Open in a separate windowFigure 1.Early steps of proposed chlorophyll breakdown pathways. MCS, Magnesium-dechelating substance.Here, we report that CLH is not involved in endogenous chlorophyll breakdown even when leaf senescence was promoted by jasmonate signaling. CLH is shown to localize to the chlorophyll-free endoplasmic reticulum (ER) and the tonoplast of intact plant cells. We found that CLH promotes the conversion of chlorophyll into chlorophyllide when leaf cells are disrupted or when CLH is genetically mislocalized to chloroplasts. To examine the possibility that plants use chlorophyll and CLH to form a binary defense system against herbivores, a generalist herbivore, Spodoptera litura larvae, was employed to investigate the toxicity of Arabidopsis leaves with genetically enhanced CLH activity and purified chlorophyllide. The results support our hypothesis, indicating plants to deploy an abundant photosynthetic pigment for defense against herbivores, which would be economic and provide adaptation benefits to plants. A potential mechanism of chlorophyllide action as part of the plant defense system is discussed based on the examination of chlorophyllide binding to the insect gut.     

Social context-dependent modification of courtship behaviour in Drosophila prolongata

Induction of alternative mating tactics by surrounding conditions, such as the presence of conspecific males, is observed in many animal species. Satellite behaviour is a remarkable example in which parasitic males exploit the reproductive investment by other males. Despite the abundance of parasitic mating tactics, however, few examples are known in which males alter courtship behaviour as a counter tactic against parasitic rivals. The fruit fly Drosophila prolongata shows prominent sexual dimorphism in the forelegs. When courting females, males of D. prolongata perform ‘leg vibration’, in which a male vibrates the female''s body with his enlarged forelegs. In this study, we found that leg vibration increased female receptivity, but it also raised a risk of interception of the female by rival males. Consequently, in the presence of rivals, males of D. prolongata shifted their courtship behaviour from leg vibration to ‘rubbing’, which was less vulnerable to interference by rival males. These results demonstrated that the males of D. prolongata adjust their courtship behaviour to circumvent the social context-dependent risk of leg vibration.